One example of a detection system is based on creation of fluorescence radiation in a sample that may be detected to analyse the sample with respect to its constitution, and an example of the use of fluorescence detection is in nucleic acid testing (NAT). This is a core element in molecular diagnostics for detecting genetic predispositions for diseases, for determining RNA expression levels or identification of pathogens, like bacteria and viruses that cause infections. Such bio-sensing methods can also be used to detect other analytes such drugs (therapeutic or abuse) or markers for disease in bodily fluids such as for example blood, urine or saliva.
The detection of fluorescence can be used both for a qualitative or a quantitative determination of the presence of a particular target analyte in a sample (e.g. DNA, protein or drug. The present invention relates to the apparatus used to detect fluorescence, and the method of use.
Many examples of chemical or biological assay methods for specific binding, capturing and even isolating such targets using for example antibodies immobilised or not are generally known form handbooks such as for example Immunology 5th edition 1998 ISBN 0723429189 see for example chapters 6, 9 29). Often used in this respect are the so called competition and sandwich assays. In a typical molecular diagnostic experiment, a bio-sample is screened for detection of certain biological components (the “target”), such as genes or proteins, the latter often providing markers for specific diseases. This is done by detecting the occurrence of selective bindings (known as hybridisation) of the target to a capture probe, such as for example an antibody. The hybridisation step is typically followed by a washing step, where all unbound target molecules are flushed away, and finally a detection step is carried out. DNA, or RNA detection is generally performed using a replication phase performed before the detection. In this replication phase the DNA or RNA to be detected and present in only small amount within the sample is replicated to larger amounts in order to facilitate reliable detection. Since, the replication step is costly in time and energy, a low detection boundary is important. The apparatus of the invention is useful in that respect.
There are two general detection approaches: homogeneous tests (in solution), and heterogeneous tests (on a surface). Heterogeneous tests are more widespread for several reasons, the most important being the fact that they allow the use of special surface sensitive techniques which yield a more sensitive detection. The detection is based on fluorescent detection of fluorescent labels attached to the target molecules. The fluorescent detection needs to be very sensitive, and for heterogeneous tests, the detection must be surface specific so as to minimize the biological background. Ideally, the fluorescent detection needs to be capable of single fluorescent label detection, while the process is kept time effective.
The capture probes can be applied in a patterned fashion which allows multiplexing (i.e. detecting many different targets in parallel). The main disadvantages of such heterogeneous, i.e. surface immobilized capture immunoassays, is that the analytes need to diffuse and bind to the surface which usually is the rate limiting step in the analysis.
Magnetic beads with surface immobilized capture probes are used frequently to extract components such as the analytes referred to above, from a solution. The beads can be pulled towards the surface by external magnets. In a second step, the beads can be re-dispersed in the fresh solution by removing the magnetic attraction. The actuation force depends on the field strength and the magnetic volume of the bead.
Magnetic beads can also be used as labels. The sensitive detection of the presence of target molecules can either be based on the signal generated by the magnetic beads (either based on optical, electrical or magnetic properties) or a signal which is generated by any other label attached to the magnetic beads.
A currently implemented solution of magnetic actuation with optical detection is the detection of the attenuation of an excitation beam entering at an oblique angle